Mechanism of trinucleotide repeat expansion by MutSβ-MutLγ and contraction by FAN1

Fig. 1. MutSβ-MutLγ cleave DNA opposite to extrahelical loops on the 5’ side. a–c A representative of three polyacrylamide gels was stained with Coomassie Brilliant blue and shows recombinant proteins used in this study. d Quantification of nicking assays with 5.6 kb-long supercoiled DNA (scDNA) and the indicated proteins. See Supplementary Fig. 1a for more details. Averages shown; error bars, s.e.m.; n = 4 independent experiments except for bars 5 and 9 (n = 3), and for bars 4 and 8 (n = 5). Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple-comparisons test. ns, non-significant. Nuclease-deficient EXO1 D173A contains the point mutation D173A. (e–g), Top, representative nuclease assays with nicked pSam_(T)4 (loop in the top strand) and the indicated proteins analyzed by Southern blotting with probes complementary to the looped strand, P1 (e), P2 (f), or to the strand opposite the loop, P4 (g). Bottom, a schematic of the DNA substrate with indicated probes. The orange triangles denote approximate DNA cleavage positions. h Densitometric profile of lanes 3 and 4 of (g). RFC and PCNA restrict DNA cleavage by MutSβ-MutLγ. i Quantification of Southern…

Fig. 2. MutSβ-MutLγ recognize diverse loops and cut DNA with a moderate sequence preference. a Nuclease assays with pSam_(T)4 or its derivatives (loop in the bottom strand) and the indicated proteins analyzed by Southern blotting with a probe complementary to the opposite looped strand, P2. Where indicated, a segment of the standard “Black” sequence of pSam was replaced with different sequences of the same length, denoted as “Red” or “Blue”. Shown are representative assays. Averages shown; error bars, s.e.m.; n = 3 independent experiments. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple-comparisons test. ns, non-significant. *, DNA not cleaved by ScaI. b Densitometric profile of lanes 3, 5 and 7 of (a). The segments containing different sequences (Black, Red, Blue) are denoted with dashed rectangles. A schematic of the DNA substrate indicates the various DNA sequences and the relative positions of the extrahelical loop and the probe. c Left, a schematic of the assay. Right, polar plot of reads from GLOE-seq performed on pSam_(T)4 with the extrahelical loop on the top strand reacted with the indicated proteins. The strand discontinuities in the looped…

Fig. 3. MutSβ, MutLγ, RPA, RFC, PCNA and Polδ catalyze repeat expansion. a A model for trinucleotide repeat expansion. b A representative of three independent DNA strand displacement assays. pSam_(CAG)4 DNA was nicked by MutSβ and MutLγ, and the reaction was then supplemented with Polδ, RPA, RFC and PCNA. Strand displacement DNA synthesis was detected by the incorporation of radioactive dCTP into high molecular weight DNA. c pSam_(CAG)4 DNA was reacted with the indicated proteins, followed by Sanger and TIDER analysis of the reaction products. While no modification of the looped strand was observed, a fraction of the opposite strand contained an insertion of the (CTG)4 sequence, supporting the model depicted in (a). The p-value associated with the trace signal is computed using a two-tailed t test, with the standard errors derived from the variance-covariance matrix. Source data are provided as a Source Data file.

Fig. 4. RFC and PCNA regulate FAN1 DNA cleavage at 3’ ends of extrahelical loops. a Top, a cartoon depicting the pulldown assay. Bottom, a representative of three pulldown experiments where FAN1 was immobilized on amylose resin using MBP-tagged FAN1, and incubated with either RFC, PCNA or both. b, c Representative nuclease assays with pSam_(CAG)4 DNA and the indicated proteins. The reaction products were analyzed by Southern blotting with probes complementary to the strand opposite the loop, P4 (b) or complementary to the looped strand, P1 (c). The red triangle indicates cleavage position at the loop site. Source data are provided as a Source Data file. d Quantification of Southern blot-based nuclease assays, such as shown in (b, c). Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test comparing FAN1 cleavage on the strand opposite the loop versus the strand that contains the loop in the presence or absence of RFC and PCNA. Averages shown; error bars, s.e.m.; n = 3 independent experiments. With RFC and PCNA, FAN1 preferentially cleaves the looped DNA strand. e Polar plot of reads obtained with GLOE-seq on pSam_(CAG)4 reacted with the…

Fig. 5. RFC, PCNA, FAN1 and Polδ excise extrahelical DNA loops. a–c Top, a representative of three independent oligonucleotide-based nuclease assays with the indicated proteins analyzed by denaturing polyacrylamide gels. Bottom, a cartoon of the used DNA substrates. The looped DNA strand was 5’-labeled, as indicated by the red asterisk (*). The DNA ends were blocked by monovalent streptavidin (STREP). Panel (a) shows the position of the initial DNA incision by FAN1-RFC-PCNA, indicated by the red triangle, which is followed by the degradation of the looped DNA by Polδ-RFC-PCNA up to the point indicated by the green triangle. Panel (b) shows that FAN1 activity can be bypassed by placing a strand discontinuity downstream of the loop, and that Polδ‘s exonuclease activity is restricted by RFC and PCNA to the DNA loop. Nuclease-deficient FAN1 ND contains D960A, D981A, R982A substitutions. Panel (c) shows that DNA incisions by FAN1-RFC-PCNA (red triangle) and Polδ-RFC-PCNA (green triangle), occur in the presence of dNTPs followed by DNA synthesis (black asterisk), leading to the synthesis of the top DNA strand without a loop. d A cartoon depicting how FAN1 and Polδ, assisted by RFC and PCNA,…

Fig. 6. FAN1 inhibits MutSβ-MutLγ through structural and catalytic functions. a Representative nicking assays with pSam_(CAG)4 DNA and the indicated proteins. Averages shown; error bars, s.e.m.; n = 4 independent experiments. Statistical analysis was performed with a two-tailed t test. Nuclease-dead FAN1 ND contains D960A, D981A, R982A substitutions. Nuclease-dead and MLH1-binding defective FAN1 MIP-MIM ND contains Y128A, F129A, L155A, L159A, D906A, D981A, R982A point mutations. FAN1 ND inhibits DNA cleavage by MutSβ-MutLγ. b Left, protein interaction assay. MutLγ (MLH1-MLH3) was immobilized using anti-MLH1 antibody, followed by incubation with MutSβ (MSH2-MSH3) and/or FAN1 variants, as indicated. MLH1-binding defective FAN1 MIP-MIM contains Y128A, F129A, L155A, L159A point mutations. When FAN1 was included (note that MBP-tagged FAN1 was used), the interaction between MutLγ and MutSβ was diminished. There was no effect on MutLγ and MutSβ interaction when MBP-tagged FAN1 MIP-MIM was used. Top, averages shown; error bars, s.e.m.; n = 3 independent experiments. The data were normalized to the interaction between MutLγ and MutSβ without FAN1. Statistical analysis was performed with a…

Fig. 7. Model for repeat expansion and contraction. Models for DNA trinucleotide repeat expansion and contraction. DNA with (CAG)n repeats is prone to the formation of secondary DNA structures (1a, b). MutLγ promotes repeat expansion both in resting cells (2) and in dividing cells (3). Specifically, MutLγ introduces biased incisions opposite the loop-out (2a and 3a), primarily oriented in the 5’ direction relative to the loop-out. In resting cells, Polδ subsequently performs displacement DNA synthesis toward the loop-out, using it as a template (2b), thus resulting in repeat expansion (2c). In proliferating cells, DNA nicks produced by MutLγ within the trinucleotide repeats (3a) can trigger replication fork collapse, resulting in one-ended DNA breaks (3b). The consequent break-induced replication and unequal sister chromatid exchange (3c) can cause abrupt changes in repeat length (3d). FAN1 has a protective function against repeat expansion (4). On one hand, FAN1 inhibits the MutSβ-MutLγ nuclease activity by disrupting the MutSβ and MutLγ complex (4a), thereby structurally preventing pathological cleavage. In addition, in conjunction with its co-factors RFC and PCNA, FAN1 nuclease…